Team:Yale/Notebook/Week12

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<h1>Week 11: July 24-31, 2011</h1>
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<ul>
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''Monday''
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<li>Monday:<ul>
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- Ran colony PCR on transformed samples; seems to confirm successful ligation (see pics below):
+
<li>Ran colony PCR on transformed samples; seems to confirm successful ligation (see pics below):</li>
-
- Picked up lyophilized cells; resuspended first in 50 mL of 50 mM Tris-HCl (pH 7.5); A280 ~ .72, A488 ~ .13; however, looked cloudy (possible aggregation?), so centrifuged at 13K rpm for 10 minutes, and UV-vised the supernatant, A280 ~ .23, A488 ~ .04, suggesting around 75% was aggregated.  Tried again to resuspend in 100 mL Tris-HCl and adding 50 mM NaCl; still no good; tried adding acid and killed protein activity
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<li>Picked up lyophilized cells; resuspended first in 50 mL of 50 mM Tris-HCl (pH 7.5); A280 ~ .72, A488 ~ .13; however, looked cloudy (possible aggregation?), so centrifuged at 13K rpm for 10 minutes, and UV-vised the supernatant, A280 ~ .23, A488 ~ .04, suggesting around 75% was aggregated.  Tried again to resuspend in 100 mL Tris-HCl and adding 50 mM NaCl; still no good; tried adding acid and killed protein activity</li>
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</ul>
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''Tuesday''
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Tuesday:<ul>
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- Miniprepped successful colony PCRs and submitted samples for sequencing
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<li>Miniprepped successful colony PCRs and submitted samples for sequencing</li>
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- Prepared more buffers for protein purification
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<li>Prepared more buffers for protein purification</li>
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- Transformed eGFP and eGFP-RiAFP and RiAFP cells
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<li>Transformed eGFP and eGFP-RiAFP and RiAFP cells</li>
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</ul></li><li>
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''Wednesday''
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Wednesday:<ul>
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- Protein purification round 2!
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<li>Protein purification round 2!</li>
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- Prepared a lysis buffer of 50 mM NaH2PO4, 300 mM NaCl (with 1 mM PMSF added before lysis), and 500 mM imidazole elution buffer (with same salts as lysis buffer)
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<li>Prepared a lysis buffer of 50 mM NaH2PO4, 300 mM NaCl (with 1 mM PMSF added before lysis), and 500 mM imidazole elution buffer (with same salts as lysis buffer)</li>
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- Innoculated eGFP, eGFP-RiAFP, and RiAFP cells
+
<li>Innoculated eGFP, eGFP-RiAFP, and RiAFP cells</li>
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- Resuspended 1 L (500 mL) worth of eGFP-RiAFP (RiAFP) pellets in around 35 mL lysis buffer
+
<li>Resuspended 1 L (500 mL) worth of eGFP-RiAFP (RiAFP) pellets in around 35 mL lysis buffer</li>
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- Sonicated (using Spiegel lab sonicator) with 1 s on/4 s off for 25 total minutes of sonication for each resuspended pellet (unforunately, RiAFP sample by itself was foaming when we returned...)
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<li>Sonicated (using Spiegel lab sonicator) with 1 s on/4 s off for 25 total minutes of sonication for each resuspended pellet (unforunately, RiAFP sample by itself was foaming when we returned...)</li>
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- Equilibrated 2 1 mL packed Ni-NTA columns (from Modis lab) with lysis buffer using peristaltic pump
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<li>Equilibrated 2 1 mL packed Ni-NTA columns (from Modis lab) with lysis buffer using peristaltic pump</li>
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- Spin-concentrated samples using
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<li>Spin-concentrated samples using millipore spinners</li>
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- Ran w FPLC/Fraction Collector Ni-NTA
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<li>Ran w FPLC/Fraction Collector Ni-NTA</li>
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</ul></li><li>
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''Thursday''
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Thursday:<ul>
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- Ran/checked gels with lots of protein
+
<li>Ran/checked gels with lots of protein</li>
-
- Grew up more cells
+
<li>Grew up more cells</li>
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- Size exclusion (small load) with eGFP-RiAFP and DTT'ed (after spin concentration)
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<li>Size exclusion (small load) with eGFP-RiAFP and DTT'ed (after spin concentration)</li>
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- Incubated with TEV
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<li>Incubated with TEV</li>
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</ul></li><li>
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''Friday''
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Friday:<ul>
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- Size exclusion of TEV-ed protein
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<li>Size exclusion of TEV-ed protein</li>
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-
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</ul></li><li>
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Saturday:<ul>
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<li>Ran gel on size-excluded protein
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</li>
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</ul>
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</ul>
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Revision as of 05:35, 28 September 2011

iGEM Yale

Week 11: July 24-31, 2011

  • Monday:
    • Ran colony PCR on transformed samples; seems to confirm successful ligation (see pics below):
    • Picked up lyophilized cells; resuspended first in 50 mL of 50 mM Tris-HCl (pH 7.5); A280 ~ .72, A488 ~ .13; however, looked cloudy (possible aggregation?), so centrifuged at 13K rpm for 10 minutes, and UV-vised the supernatant, A280 ~ .23, A488 ~ .04, suggesting around 75% was aggregated. Tried again to resuspend in 100 mL Tris-HCl and adding 50 mM NaCl; still no good; tried adding acid and killed protein activity
    Tuesday:
    • Miniprepped successful colony PCRs and submitted samples for sequencing
    • Prepared more buffers for protein purification
    • Transformed eGFP and eGFP-RiAFP and RiAFP cells
  • Wednesday:
    • Protein purification round 2!
    • Prepared a lysis buffer of 50 mM NaH2PO4, 300 mM NaCl (with 1 mM PMSF added before lysis), and 500 mM imidazole elution buffer (with same salts as lysis buffer)
    • Innoculated eGFP, eGFP-RiAFP, and RiAFP cells
    • Resuspended 1 L (500 mL) worth of eGFP-RiAFP (RiAFP) pellets in around 35 mL lysis buffer
    • Sonicated (using Spiegel lab sonicator) with 1 s on/4 s off for 25 total minutes of sonication for each resuspended pellet (unforunately, RiAFP sample by itself was foaming when we returned...)
    • Equilibrated 2 1 mL packed Ni-NTA columns (from Modis lab) with lysis buffer using peristaltic pump
    • Spin-concentrated samples using millipore spinners
    • Ran w FPLC/Fraction Collector Ni-NTA
  • Thursday:
    • Ran/checked gels with lots of protein
    • Grew up more cells
    • Size exclusion (small load) with eGFP-RiAFP and DTT'ed (after spin concentration)
    • Incubated with TEV
  • Friday:
    • Size exclusion of TEV-ed protein
  • Saturday:
    • Ran gel on size-excluded protein

Retrieved from "http://2011.igem.org/Team:Yale/Notebook/Week12"